Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Use of mTRAQ derivatization reagents on tissues for imaging neurotransmitters by MALDI imaging mass spectrometry: the triple spray method.

During drug development, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is used for visually elucidating the distribution of substances such as biomarkers, candidate compounds, and metabolites in the tissues. However, it is difficult to make relative comparisons between tissue sections and there are still many challenges. Here, we report a new method of "triple spray" for the comparison of analyte distribution in multiple tissue slices. This method targets amino acids and amines, and it incorporates the application of the internal standard in the on-tissue derivatization step. With further development, it has the potential to alleviate problems caused by the matrix effect. Initially, we measured three serial sections of rat brain to verify the efficacy of this method. In the hypothalamus, where gamma-aminobutyric acid (GABA) is known to be present in high concentration, the GABA levels of the three serial section showed little variation (CV = 1.62%). Subsequently, we compared the GABA level in the brain between stroke-prone spontaneous hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats with three individuals each. It showed significant differences between these models at the pre-selected region of interest (p < 0.05). Our results show that the triple spray allows for relative comparison among multiple tissue slices with high reproducibility. Graphical abstract.

Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor.

Aberrant production of proinflammatory cytokines is linked to many autoimmune diseases, and their inhibition by small molecule compounds is considered beneficial. Here, we performed phenotypic screening in IFNγ/LPS-activated RAW264.7, mouse macrophage cells, and discovered AS2677131 and AS2795440 as novel and potent inhibitors of IL-12p40, a subunit of IL-23. Interestingly, these compounds exhibited unique pharmacological activities in their inhibition of the production of IL-12p40, IL-6 and IL-1ß but not TNFα in activated macrophages or dendritic cells, and expression of IgM-induced MHC class II on B cells. To reveal these mechanisms, we synthesized two different activity probes which were structurally related to the AS compounds, and identified probe-specific binding proteins, including PIKfyve, a Class III PI kinase. The AS compounds inhibited PIKfyve activity and mimicked the properties of PIKfyve-deficient cells, eventually validating PIKfyve as target molecule. Regarding mechanism, AS2677131 regulated DNA binding activity of c-Rel on IL-12p40 and IL-1ß promoter. As expected, a PIKfyve inhibitor prevented the development of arthritis in rats. Taken together, our findings of the novel and potent PIKfyve inhibitors AS2677131 and AS2795440 reveal the critical role of PIKfyve in proinflammatory cytokine production and B cell activation, and may indicate a potential new therapeutic option for treatment of inflammatory diseases.

Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) complex, which is required for survivin expression.

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Interleukin enhancer-binding factor 3/NF110 is a target of YM155, a suppressant of survivin.

Survivin is responsible for cancer progression and drug resistance in many types of cancer. YM155 selectively suppresses the expression of survivin and induces apoptosis in cancer cells in vitro and in vivo. However, the mechanism underlying these effects of YM155 is unknown. Here, we show that a transcription factor, interleukin enhancer-binding factor 3 (ILF3)/NF110, is a direct binding target of YM155. The enhanced survivin promoter activity by overexpression of ILF3/NF110 was attenuated by YM155 in a concentration-dependent manner, suggesting that ILF3/NF110 is the physiological target through which YM155 mediates survivin suppression. The results also show that the unique C-terminal region of ILF3/NF110 is important for promoting survivin expression and for high affinity binding to YM155.

Identification and relative quantitation of an orphan G-protein coupled receptor SREB2 (GPR85) protein in tissue using a linear ion trap mass spectrometer.

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.

Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

Increased concentration of neutrophil elastase in urine from patients with interstitial cystitis.

OBJECTIVE: To identify proteins associated with interstitial cystitis (IC), protein profiles were analyzed using a proteomics-based approach. The study tested whether neutrophil elastase in urine correlates with the symptomatic condition of IC. MATERIAL AND METHODS: Proteins in urine from IC patients and healthy subjects were analyzed through a comparative proteomics approach using two-dimensional difference in-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Neutrophil elastase activity was measured by the digestion of peptide substrate. RESULTS: The urinary neutrophil elastase concentration was significantly higher in IC patients with pain than in healthy subjects. It was significantly increased in patients with small bladder capacity (median 6.31 ng/ml in IC with a bladder capacity < 200 ml vs 1.15 ng/ml in IC with a bladder capacity > or = 200 ml and 0.18 ng/ml in healthy bladders, p < 0.01). The concentration of neutrophil elastase did not correlate with the neutrophil count in the urine of IC patients. CONCLUSION: The concentration of neutrophil elastase increased in the urine of the IC patient subset with bladder pain and small bladder capacity.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Absence of increased alpha1-microglobulin in IgA nephropathy proteinuria.

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.

YM-216391, a novel cytotoxic cyclic peptide from Streptomyces nobilis. II. Physico-chemical properties and structure elucidation.

YM-216391, a novel cytotoxic cyclic peptide, has been isolated from the cultured mycelium of Streptomyces nobilis JCM 4274. The planar structure of YM-216391 was assigned on the basis of 1D and 2D NMR spectroscopic techniques. The absolute configuration of the amino acid residues in YM-216391 was determined by Marfey's analysis and chiral HPLC analysis of its acid hydrolysate.